JOURNAL OF NEUROINFLAMMATION

Perriard et al. Journal of Neuroinflammation (2015) 12:119 DOI 10.1186/s12974-015-0335-3

RESEARCH Open Access

Interleukin-22 is increased in multiplesclerosis patients and targets astrocytes

Guillaume Perriard1, Amandine Mathias1, Lukas Enz3, Mathieu Canales1, Myriam Schluep2, Melanie Gentner3,Nicole Schaeren-Wiemers3 and Renaud A. Du Pasquier1,2*

Abstract

Background: Increasing evidences link T helper 17 (Th17) cells with multiple sclerosis (MS). In this context,interleukin-22 (IL-22), a Th17-linked cytokine, has been implicated in blood brain barrier breakdown and lymphocyteinfiltration. Furthermore, polymorphism between MS patients and controls has been recently described in the genecoding for IL-22 binding protein (IL-22BP). Here, we aimed to better characterize IL-22 in the context of MS.

Methods: IL-22 and IL-22BP expressions were assessed by ELISA and qPCR in the following compartments of MSpatients and control subjects: (1) the serum, (2) the cerebrospinal fluid, and (3) immune cells of peripheral blood.Identification of the IL-22 receptor subunit, IL-22R1, was performed by immunohistochemistry and immunofluorescencein human brain tissues and human primary astrocytes. The role of IL-22 on human primary astrocytes was evaluatedusing 7-AAD and annexin V, markers of cell viability and apoptosis, respectively.

Results: In a cohort of 141 MS patients and healthy control (HC) subjects, we found that serum levels of IL-22 weresignificantly higher in relapsing MS patients than in HC but also remitting and progressive MS patients. Monocytes andmonocyte-derived dendritic cells contained an enhanced expression of mRNA coding for IL-22BP as compared to HC.Using immunohistochemistry and confocal microscopy, we found that IL-22 and its receptor were detected onastrocytes of brain tissues from both control subjects and MS patients, although in the latter, the expression was higheraround blood vessels and in MS plaques.Cytometry-based functional assays revealed that addition of IL-22 improved the survival of human primary astrocytes.Furthermore, tumor necrosis factor α-treated astrocytes had a better long-term survival capacity upon IL-22co-treatment. This protective effect of IL-22 seemed to be conferred, at least partially, by a decreased apoptosis.

Conclusions: We show that (1) there is a dysregulation in the expression of IL-22 and its antagonist, IL-22BP, inMS patients, (2) IL-22 targets specifically astrocytes in the human brain, and (3) this cytokine confers an increasedsurvival of the latter cells.

Keywords: Multiple sclerosis, Interleukin-22, Astrocytes, Survival

BackgroundMultiple sclerosis (MS) occurs in genetically predisposedyoung adults with probable environmental triggers [1].Infiltrating auto-reactive immune cells, in synergy withresident glial cells, will cause neuroinflammation andneurodegeneration, as characterized by demyelination,

* Correspondence: renaud.du-pasquier@chuv.ch1Laboratory of Neuroimmunology, Center of Research in Neurosciences,Department of Clinical Neurosciences and Service of Immunology andAllergy, Department of Medicine, CHUV, 1011 Lausanne, Switzerland2Service of Neurology, Department of Clinical Neurosciences, CHUV BH-10/131, 46, rue du Bugnon, 1011 Lausanne, SwitzerlandFull list of author information is available at the end of the article

© 2015 Perriard et al. This is an Open Access a(http://creativecommons.org/licenses/by/4.0),provided the original work is properly creditedcreativecommons.org/publicdomain/zero/1.0/

axonal loss, and finally irreversible damage to the centralnervous system (CNS) [2]. Pro-inflammatory T helper17 (Th17) cells have been associated with MS [3–9], butthe function of Th17 cells in the pathogenesis of MS isstill a matter of debate [10–12].Interleukin-22 (IL-22), a Th17-linked cytokine, is associ-

ated with autoimmune diseases such as inflammatorybowel diseases and psoriasis [13]. Depending on the tar-geted tissue and the cytokine milieu in which it is released,IL-22 can contribute to inflammation, chemotaxis, andhost defense but also to cell survival, tissue protection,wound healing, and epithelial cell proliferation [14–20].

rticle distributed under the terms of the Creative Commons Attribution Licensewhich permits unrestricted use, distribution, and reproduction in any medium,. The Creative Commons Public Domain Dedication waiver (http://) applies to the data made available in this article, unless otherwise stated.

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IL-22 modulates immunity at barrier surface in multiplehuman diseases [21]. Together with IL-17, IL-22 seemsto compromise the blood brain barrier integrity, enab-ling lymphocyte ingress into the CNS, which raises thepossibility that this cytokine may contribute to MSseverity [22]. The melanoma cell adhesion molecule(MCAM) has been associated with infiltration of T cellsinto CNS lesions together with an increased expressionof IL-17 and IL-22 in MCAM+ T cells as compared toMCAM− T cells [23, 24]. Somewhat contrasting with theprevious findings, some data suggest that IL-22 may notnecessarily be pro-inflammatory: in Theiler’s virus-induced demyelination in mice, epitope-specific CD8+ Tcells causing minimal cytotoxicity in the CNS expressed ahigher level of IL-22 mRNA than highly cytotoxic CD8+ Tcells [25]. IL-22 may even be useful for tissue-protectivetherapy [26]. Further suggesting that IL-22 may be in-volved in MS, genetic studies showed that the gene codingfor IL-22 binding protein (IL-22BP, also called IL-22RA2),an antagonist of IL-22 [27–30], harbored different singlenucleotide polymorphism in MS patients as compared tocontrol subjects [31–34]. Interestingly, this secreted IL-22 inhibitory receptor exacerbates experimental auto-immune encephalomyelitis (EAE) disease course [31, 35],raising the question whether IL-22 itself may have an anti-inflammatory function in EAE.These data suggest that IL-22 may be involved in the

immunopathogenesis of MS. However, this cytokine hasbeen barely studied in MS patients. Here, we investi-gated whether IL-22 and IL-22BP are dysregulated inMS. We further aimed at identifying its target cells inhuman brain tissues, in particular in MS patients, anddetermining its functional effect in the CNS.

MethodsStudy subjectsFor the studies pertaining to the determination of IL-22and IL-22BP levels in the blood, 141 MS patients andhealthy control (HC) subjects were enrolled and dividedinto subgroups according to the disease type (Table 1).The diagnosis of MS followed the revised McDonald cri-teria [36]. The category of clinically active multiple scler-osis patients comprised relapsing remitting (RR)-MS orclinically isolated syndrome (CIS), who had a relapsethat started less than 2 months prior to our assays. Thecategory of clinically inactive multiple sclerosis patientsincluded RR-MS and CIS patients who were in remis-sion, as defined by an interval of more than 3 monthsafter the last relapse. The category of progressive MS pa-tient group contained patients with secondary progres-sive (SP)-MS or primary progressive (PP)-MS. Clinicallyinactive, SP- and PP-MS patients were not under anytreatment within the 3 months prior to the blood draw.Among the 26 clinically active MS patients, four were

on interferon-β, one on natalizumab, and one on fingoli-mod treatments. None of the MS patients had receivedcorticosteroids in the previous 3 months. All enrolledpatients and healthy control subjects signed an informedconsent form, according to our institution review board.For immunohistochemistry studies, brain biopsies

from 11 non-MS patients were obtained from neurosur-gical resections performed in the service of neurosurgeryat the CHUV in Lausanne, hereafter referred to as the“Lausanne cohort” (Table 2). Tissues from five MS pa-tients with their seven control counterparts obtainedafter postmortem autopsies were processed in Basel andnamed “Basel cohort” (Table 2). All human brain tissueswere collected in accordance with local ethical commit-tee from the University Hospitals of Lausanne and Baseland the UK MS Tissue Bank.

Human brain tissuesLausanne cohortIn Lausanne, biopsied brain tissues were obtained onlyfrom non-MS study subjects. Just after biopsy, theseex vivo brain tissues, encompassing either white matter(WM), gray matter (GM), or both, were frozen and storedat −80 °C until they were cut to obtain 12-μm cryosectionsfor immunofluorescence experiments. Hereafter, biopsiedbrain tissues from Lausanne cohort are named L-C1 toL-C11 (Lausanne-Control #1–11; Table 2).

Basel cohortIn Basel, brain tissues were obtained from postmortemautopsies supplied by the UK Multiple Sclerosis TissueBank at the Imperial College (UK Multicentre ResearchEthics Committee, MREC/02/2/39), funded by the Mul-tiple Sclerosis Society of Great Britain and NorthernIreland (registered charity 207,495). In addition to thebrains of MS patients, there were also brain samples, in-cluding cortex and subcortical WM, from non-MS con-trol patients (Table 2). Postmortem autopsy tissues weredirectly frozen and stored at −80 °C before use. Cryostattissue sections (12 μm) from MS and control subjectwere mounted on Superfrost plus slides (Merck), driedfor 30 min, and fixed with 4 % paraformaldehyde inphosphate-buffered saline (PBS) for 10 min at roomtemperature (RT). Slides were washed in PBS beforestaining. Brain tissues from Basel cohort are listed B-C1to B-C7 and B-MS1 to B-MS5, referring to Basel controlsubjects and MS patients, respectively (Table 2).

Primary human astrocytesHuman primary astrocytes (HA) derived from braincerebral cortex were purchased from ScienCell ResearchLaboratory and were cultured according to the pro-vider’s instructions. Briefly, HA were grown and culturedat 37 °C with 5 % CO2, in astrocyte medium (AM),

Table 1 Study subjects for the assessment of IL-22 and IL-22BP in the blood

Category N = 141 Male/femaleratio

Age at blood draw(years)a

EDSSa Disease duration(years)a

Last relapse(months)a

Clinically active MS patients 26 7/19 31.5 ± 10.0 2.00 ± 1.00 0.63 ± 7.19 0.46 ± 0.82

Clinically inactive MS patients 35 10/25 39.5 ± 11.5 1.50 ± 0.63 7.00 ± 9.25 16.87 ± 34.43

Progressive MS patients 35 12/23 52.0 ± 15.0 4.50 ± 2.50 17.0 ± 12.0 –

Healthy control subjects 45 21/24 34.0 ± 28.5 – – –

MS multiple sclerosis, EDSS expanded disability status scaleaMedian ± interquartile range

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supplemented with 2 % fetal calf serum (FCS) and 1 %astrocyte nutritive supplement with 1 % penicillin/strepto-mycin (referred as “complete astrocyte medium”). For im-munofluorescence, cells were fixed for 15 min with 4 %paraformaldehyde and stored in phosphate-buffered saline(PBS) at 4 °C. For flow cytometry, cells were resuspendedwith trypsin (BioConcept) and first labeled with LIVE/DEAD fixable violet dead cell stain (Life Technologies).Then, HA were stained with cytofix/cytoperm (BD Biosci-ences) with mouse anti-IL-22R1 (clone 305405, R&D Sys-tems)/mouse IgG1 isotype control (clone 11711, R&DSystems) or rabbit anti-IL-10R2 (sc-69580, Santa CruzBiotechnology)/rabbit IgG isotype control (AB-105-C,R&D Systems) primary antibodies and followed by donkeyanti-mouse IgG AF546 and goat anti-rabbit IgG AF488(Invitrogen) secondary antibodies. Alternatively, cell sus-pension was directly used for staining as described in“Proliferation and cell death/apoptosis assays” section.Cells were processed with an LSRII flow cytometer (BDBiosciences) and were analyzed with FlowJo software (ver-sion 9.1.11, Treestar).

PBMC isolation and cell sortingPeripheral blood mononuclear cells (PBMC) were freshlyisolated by Ficoll (GE Healthcare Biosciences) as de-scribed previously [37]. PBMC subpopulations weresorted with anti-CD4, anti-CD8, anti-CD14, anti-CD19,and anti-CD56 MicroBeads (Miltenyi Biotec) with anautoMACS Pro Separator (Miltenyi Biotec) according tomanufacturer’s instructions. The purity of sorted cellswas checked by flow cytometry with the following anti-bodies: anti-CD4 APC-H7 (clone SK3, BD Biosciences),anti-CD4 ECD (clone SFCI12T4D11, Beckman Coulter),anti-CD8 FITC (clone RPA-T8, BD Biosciences), anti-CD11c APC (clone B-ly6, BD Biosciences), anti-CD14PB (clone M5E2, BD Biosciences), anti-CD19 PE (cloneHIB19, eBioscience), and anti-CD56 PE-Cy7 (cloneNCAM16.2, BD Biosciences) on a LSRII flow cytometer(BD Biosciences). We did not pursue the proposedexperiments if the purity of sorted cells was less than 90%. Analyses were performed using FlowJo software(Treestar).

Generation of in vitro monocyte-derived dendritic cellsSorted CD14+ cells were incubated for 6 days at 1*10e6cells per ml in Roswell Park Memorial Institute (RPMI;Gibco, Life Technologies) supplemented with 10 % FCS(heat inactivated, PAA Laboratories), 50 ng/ml premiumgrade recombinant granulocyte macrophages colony-stimulating factor (GM-CSF) and 20 ng/ml premiumgrade recombinant IL-4 (Miltenyi Biotec) to obtain dif-ferentiated monocyte-derived DCs (moDCs). Cell differ-entiation quality was checked by flow cytometry withthe following antibodies: anti-CD11c APC (clone B-ly6,BD Biosciences) and anti-CD14 PB (clone M5E2, BDBiosciences). Proper differentiation was considered ascompleted when at least 80 % of the harvested cells wereCD11c+CD14−. For mRNA analysis, moDCs were lysedwith RLT Plus buffer (Qiagen) and kept at −20 °C untilfurther extraction.

Leukocyte stimulationWhole PBMC were left untreated or stimulated with 100ng/ml staphylococcal enterotoxin B (SEB, Sigma) at2*10e6 cells per ml for 18 h at 37 °C. Supernatants wereharvested and stored at −80 °C until use. CD4+, CD8+,CD14+, CD19+, CD56+ sorted cells, and moDCs wereeither left untreated or stimulated at 2*10e6 cells per mlwith 100 ng/ml SEB, 1 μg/ml resiquimod (R848) (Invivo-Gen)—a toll-like receptor 7 and 8 ligand, a potent acti-vator of both monocytes and B cells—, 10 μ/ml CD3/28beads (Gibco, Life Technologies) for 18 h or 100 ng/mlphorbol myristate acetate (PMA, Sigma) and 1 μg/mlionomycin (iono, Sigma) for 6 h at 37 °C.For mRNA analysis, cells were lysed with RLT Plus

buffer (Qiagen) and kept at −20 °C until furtherextraction.

ELISAMeasurement of IL-22 in the serum, cerebrospinal fluid(CSF), or supernatant of stimulated PBMC was performedwith the Human IL-22 ELISA Ready-SET-Go (eBioscience)according to manufacturer’s instructions and read withOpsys MR (Dynex International) instrument.

Table 2 Study subjects for the assessment of IL-22 and IL-22 receptor in the brain

Patient Gender Age at surgery(years)

Type ofsurgery

Neuropathology reported fromautopsy

Cause of surgery/death

Postmortemtime (hours)

MS type Disease duration(years)

Lausanne cohort

L-C1 F 60 Biopsy – Cerebellar softening – – –

L-C2 M 31 Biopsy – Epilepsy – – –

L-C3 F 51 Biopsy – Hematoma – – –

L-C4 M 43 Biopsy – Aneurysm – – –

L-C5 M 41 Biopsy – Hematoma – – –

L-C6 n/a n/a Biopsy – Polymorphicneuroectodermaltumor

– – –

L-C7 n/a n/a Biopsy – Glioblastoma – – –

L-C8 M 32 Biopsy – Cavernoma – – –

L-C9 F 53 Biopsy – Epilepsy – – –

L-C10 M 51 Biopsy – Malformation – – –

L-C11 M 39 Biopsy – Epilepsy – – –

Basel cohort

B-C1 M 75 Autopsy Many documented neuropathologicalfindings

Cerebrovascularaccident, aspirationpneumonia

17 – –

B-C2 M 64 Autopsy Occasional hypoxic neurons,perineuronal oedema, stasis oferythrocytes in vessels, manyleukocyte infiltrates

Cardiac failure 18 – –

B-C3 M 84 Autopsy Fibrosis of vessel walls, mild WMgliosis, perivascular oedema

Bladder cancer,pneumonia

5 – –

B-C4 M 35 Autopsy – Carcinoma of thetongue

22 – –

B-C5 M 64 Autopsy Occasional hypoxic nerve cells,perineuronal oedema, fibrosis of themeninges

Cardiac failure 18 – –

B-C6 F 84 Autopsy Old cortical microinfarcts and acuteglobal hypoxic changes. Senilechanges are also present (amyloiddeposits)

Congestive cardiacfailure, ischemic heartdisease, atrialfibrillation

24 – –

B-C7 F 60 Autopsy Brain with diffuse hypoxic changes Ovarian cancer 13 – –

B-MS1 M 40 Autopsy No lesion, few leukocyte infiltrates Respiratory failure,sepsis, MS

10 SP-MS 9

B-MS2 F 78 Autopsy No lesion, some vessels withleukocyte infiltrates

Metastatic carcinomaof bronchus

5 SP-MS 42

B-MS3 F 34 Autopsy Lesions in GM and WM, leukocytesaround vessels

Pneumonia 12 SP-MS n/a

B-MS4 F 49 Autopsy Lesions in WM, leukocytes infiltrates Bronchopneumonia,MS

7 SP-MS 21

B-MS5 M 44 Autopsy n/a Bronchopneumonia 16 SP-MS n/a

M male, F female, C control, MS multiple sclerosis, B Basel, L Lausanne, GM gray matter, n/a not available, SP secondary progressive, WM white matter

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IL-22BP was measured in the serum and CSF by ahome-made kit. “Maxisorp Immunoplates” 96-wellplates (Nunc) were coated with coating solution (15 mMNa2CO3, 34.8 mM NaHCO3) mixed with goat anti-IL-22BP antibody (AF1087, R&D Systems) diluted 1:500and incubated overnight at 4 °C. The next day, blocking

was performed by filling 200 μl/well PBS containing 0.05% Tween 20 and 1 % bovine serum albumin (BSA) (PBS/T-1 % BSA) (Sigma) with 2 h incubation at 37 °C. Afterthree washes with wash buffer solution (BD Biosciences),100-μl standard dilutions (1087-BP-025, R&D Systems)and samples diluted with 50 μl PBS/T-1 % BSA were

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added to each well and incubated overnight at 4 °C. Thethird day, plates were washed three times and 50 μl/wellof rabbit anti-IL-22BP (sc-134974, Santa Cruz Biotech-nology) diluted 1:200 in PBS/T-1 % BSA were added and1 h incubation at 37 °C was performed. Following threewashes, addition of 50 μl of mouse anti-rabbit biotinyl-ated antibody (550346, BD Biosciences) diluted 1:3,000in PBS/T-1 % BSA was performed and the plates wereincubated 1 h at 37 °C. After another round of threewashes, 50 μl/well of 1:200 streptavidin-HRP (DY998,R&D Systems) in PBS/T-1 % BSA was added and incu-bated at RT for 30 min. Finally, after six washing steps,ELISA was revealed and developed with 100 μl/wellrevelation buffer (DY999 R&D Systems) in a 20-min in-cubation period at RT, protected from light. Reactionwas stopped with 50 μl/well 1 M H2SO4, and plates wereread at 450 nm with Opsys MR (Dynex International)device. Detection limit was set at 0.5 ng/ml to fully guar-antee specificity and reliability of positive samples, basedon data of the recombinant IL-22BP standard curve(R&D Systems).

RNA extraction, reverse transcription and quantitativePCRThe biological material was lysed with RLT Plus buffer(Qiagen) and stored at −20 °C until RNA extraction. TheRNA isolation was performed with the RNeasy PlusMini kit (Qiagen). Up to 0.5-μg purified RNA (concen-tration measured with a NanoDrop 2000, Thermo Scien-tific) was taken for reverse transcription utilizing theQuantitect Reverse Transcription kit (Qiagen). Quantita-tive PCR was performed with the QuantiTect SYBRgreen PCR mix (Qiagen) and QuantiTect primer assaysfor 18S ribosomal RNA, IL-22BP set (Qiagen). Micro-Amp Fast Optical 96-well reaction plate (Applied Biosys-tems, Life Technologies) was run with a StepOnePlusReal-Time PCR System (Applied Biosystems, Life Tech-nologies). The relative expression of each sample wascalculated with the “2e(−ΔCt)” equation where the Ct isdefined as the cycle number at which the SYBR greenfluorescence crosses the threshold (arbitrary set andfixed for all experiments performed) and the Δ is the dif-ference between the Ct of the sample tested and thehousekeeping gene Ct. Melting curve analysis was per-formed to ensure reaction specificity.

ImmunohistochemistryFor immunohistochemistry staining, tissue sections weretreated with 0.6 % hydrogen peroxide in 80 % methanolfor 30 min to inactivate endogenous peroxidase andblocked with blocking buffer (1 % normal donkey serum,0.1 % Triton, 0.05 % Tween) for 1 h. The tissue sectionsfor myelin oligodendrocyte glycoprotein (MOG) werethen additionally defatted in 100 % methanol for 8 min

at −20C°. Sections were incubated with following pri-mary antibodies overnight at 4 °C: mouse anti-MOG(clone Z12, kindly provided by R. Reynolds) to targetmyelin, mouse anti-IL-22R1 (clone 305405, R&D Sys-tems)/mouse IgG1 isotype control (clone 11711, R&DSystems), rabbit anti-glial fibrillary acidic protein (GFAP)(AB5804, Millipore; Z0334, DakoCytomation) to labelastrocytes, and rabbit anti-Caveolin-1 (Cav-1) (N-20,Santa Cruz Biotechnology) for endothelia staining/rabbitIgG isotype control (AB-105-C, R&D Systems; 12–370,Millipore). Secondary biotinylated antibodies (VectorLaboratories) were applied for 1 h at room temperature,together with 4′,6-diamidino-2-phenylindole (DAPI,Invitrogen Life Technologies) counterstaining, followedby avidin-biotin complex reagent (Vector Labs) for 30min. Color reaction was performed with 3-amino-9-ethylcarbazole. Cells were stained in hematoxylin for 5min and rinsed afterwards under running tap water.Image acquisition was performed with a Zeiss Axiovision(Carl Zeiss Microscopy) microscope, and picture analysiswas performed with Axiovision software (versionV4.8.1.0, Carl Zeiss Microscopy).

Laser scanning confocal microscopyImmunostainings were performed with the followingprimary antibodies: goat anti-IL-22 (AF782, R&D Sys-tems)/goat IgG (AB-108-C, R&D Systems), mouse anti-IL-22R1 (clone 305405, R&D Systems)/mouse IgG1

(clone 11711, R&D Systems), rabbit anti-IL-10R2 (sc-69580, Santa Cruz Biotechnology), rabbit anti-GFAP(AB5804, Millipore; Z0334, DakoCytomation), mouseanti-GFAP-Cy3 (for HA only, clone G-A-5, Sigma),rabbit anti-Caveolin-1 (N-20, Santa Cruz Biotechnol-ogy)/rabbit IgG (AB-105-C, R&D Systems; 12–370,Millipore), sheep anti-von Willebrand factor (VWF)(GTX74137, GeneTex) for vessel labeling, and chickenanti-microtubule-associated protein (MAP)-2 (ab5392,Abcam) to target neurons/chicken IgG (GTX35001,GeneTex) and with the following secondary antibodies:donkey anti-goat IgG AF488, donkey anti-mouse IgGAF546 and AF647, goat anti-rabbit IgG AF488, donkeyanti-rabbit IgG AF647, goat anti-chicken IgG AF647,goat anti-sheep AF647 (Invitrogen) and, finally, donkeyanti-rabbit IgG AF488 (Jackson ImmunoResearch) anddonkey anti-rabbit IgG CSL467 (Santa Cruz Biotechnol-ogy). To reduce autofluorescence, tissue sections of theBasel cohort were incubated in cupric sulfate in ammo-nium buffer (10 mM CuSO4, 50 mM CH3COONH3, pH5.0) for 30 min before secondary antibody staining. Nu-clei staining was done with DAPI (Invitrogen Life Tech-nologies). Slices were mounted with Vectashield (VectorLaboratories). Image acquisition was done with a ZeissLSM 710 Quasar (Carl Zeiss Microscopy) confocal, andpicture analysis was performed with Zeiss ZEN 2009

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(Carl Zeiss Microscopy), ImageJ (version 1.46r, NationalInstitutes of Health), and Axiovision softwares (versionV4.8.1.0, Carl Zeiss Microscopy). Images were taken,and post-acquisition processing (brightness and con-trast) was done the same way for specific antibodies andtheir isotype controls.

Proliferation and cell death/apoptosis assaysFor functional experiments, cells were cultured at lowpassage (three to six passages) in complete astrocytemedium, prior to transfer in 24-well plates (Costar) at20,000 cells/well in RPMI only medium, 300 μl/well, onday −1. On day 0 and then every other day over a 9-dayperiod, HA were treated with six different conditions:astrocyte medium without FCS as reference medium;RPMI only (referred as “untreated” or negative control) asstandard minimal medium to provide only essential nutri-ent to the cells; IL-22 at 50 ng/ml (R&D Systems); tumornecrosis factor (TNF)α at 10 ng/ml (R&D Systems); TNFαand IL-22 co-treatment; and finally, 100 nM staurosporine(STS, Sigma) as positive control to induce apoptosis andcell death. TNFα was chosen as a pro-inflammatory cyto-kine considering its paramount role in MS [38].To assess for cell proliferation, carboxyfluorescein suc-

cinimidyl ester (CFSE, Biolegend) staining was done atthe beginning of the kinetic (day −1), such as alreadyperformed in the lab [39], whereas to assess for celldeath and apoptosis, staining with 7-aminoactinomycinD (7-AAD, BD Biosciences) and Annexin V AF647 (Invi-trogen Life Technologies) in Annexin-binding buffer(Invitrogen Life Technologies), respectively, were per-formed at day 1, 2, 3, 4, 5, 7, and 9, according to manu-facturer’s instructions. Annexin V marker analysis wasperformed on 7-AAD− pregated cells. Samples were runwith an LSRII flow cytometer (BD Biosciences) and wereanalyzed with FlowJo software (version 9.1.11, Treestar).

Statistical analysisStatistical analyses were performed with GraphPad Prismsoftware (version 6.04, GraphPad Software). The differ-ences among groups (three or more) were first testedusing Kruskal-Wallis test for multiple non-normally dis-tributed variables. Unpaired non-parametric two-tailedMann–Whitney was used to test groups two-by-two. AP value < 0.05 was considered significant.

ResultsIncreased IL-22 in active MS patientsFirst, using ELISA, we found that there was a strongtrend (p = 0.07) for an increase of IL-22 protein in theserum of 63 MS patients as compared to 13 HC (Fig. 1a).Interestingly, the level of IL-22 in the serum of MS pa-tients with active disease was higher than in the serumof inactive (p = 0.017) and progressive (p = 0.015) MS

patients and, especially, of HC (p = 0.003) (Fig. 1b). IL-22 was not detectable in the CSF of patients with activeMS (Fig. 1c). Of note, no lumbar puncture was per-formed in the other categories of study patients.Next, we found that the supernatant of SEB-stimulated

PBMC of 74 MS patients secreted a higher amount ofIL-22 than of 32 HC (p = 0.0436, Fig. 1d), a findingwhich was ascribable to the active category of MS pa-tients (active versus HC: p = 0.0048, active versus in-active: p = 0.0216, Fig. 1e). Then, we investigated whichleukocyte subtypes secreted IL-22. We found that CD4+

T cells accounted for most of the production of IL-22;nevertheless, and as already reported, monocytes, B cells,CD8+ T cells, and natural killer (NK) cells were also ableto produce and secrete significant amount of IL-22(Additional file 1: Figure S1) [40]. Of note, unstimulatedPBMC released a low, but not null, level of IL-22, con-sistent with previous reports [41]. Therefore, we testedseveral polyclonal stimulations (SEB, R848, PMA/iono-mycin, and CD3/CD28 beads), and all showed similar ef-ficacy, except for R848 which was induced much less IL-22 secretion from CD4+ T cells than other stimulants(Additional file 1: Figure S1).To further examine the putative implication of IL-22

in MS, we looked at its soluble antagonist, i.e., IL-22BP.Indeed, IL-22BP gene polymorphism has been recentlyassociated with MS [32]. Looking first at the proteinlevel, we did not find a difference in terms of IL-22BPprotein in the sera of 63 MS patients versus 13 healthycontrols (HC); however, there was a trend (p = 0.14) to-wards an increased secretion of IL-22BP in MS patientsas compared to HC (Fig. 1f ). Those 76 study subjectsand patients were the very same who were tested for thecontent of IL-22 in the serum (see above). Some MS pa-tients harbored high levels of soluble IL-22BP, reachinglevels of 10 ng/ml and more (Fig. 1f–g); however, therewas no difference between the categories of MS patients(Fig. 1g). Interestingly, IL-22BP was detected in the CSFof 13/15 active MS patients who had a lumbar punctureat the same time as this assay (Fig. 1h).Then, we found that among different sorted subpopu-

lations of blood immune cells, CD14+ monocytes and,especially, in vitro differentiated moDCs contained thehighest levels of mRNA coding for IL-22BP (Additionalfile 2: Figure S2), confirming previous literature data[31, 35, 42, 43]. We found an increased expression of IL-22BP mRNA in the monocytes of 51 patients as com-pared to 30 HC (p = 0.016; Fig. 1i) and in the moDCs ofa subset of 15 MS patients as compared to 10 HC(p = 0.016; Fig. 1k). Interestingly, although there was nodifference between the categories as a whole (p = 0.108with the Kruskal-Wallis test), the higher expression ofIL-22BP mRNA in the monocytes of MS patientsseemed to be mostly accountable to the category of

Fig. 1 (See legend on next page.)

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(See figure on previous page.)Fig. 1 IL-22BP and IL-22 are increased in MS patients as compared to healthy controls. The IL-22 and IL-22BP expressions were assessed by ELISA(a–h) and qPCR (i–k) in the serum (a, b and f, g), CSF (c, h), isolated monocytes (i, j), and moDCs (k) and supernatant of SEB-stimulated PBMCfor 18 h (d, e) isolated from MS patients and healthy controls. Each dot represents a patient. The bars represent the median. Dashed red linesrepresent the detection limit. Active: clinically active MS patients, inactive: clinically inactive MS patients, progressive: primary and secondaryprogressive MS patients. Differences among the four groups were significant with Kruskal-Wallis test (b, e). Unpaired non-parametric Mann–Whitneytest was used to compare groups two-by-two. *P < 0.05, **P < 0.01

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active MS (active MS patients versus HC: p = 0.037,Fig. 1j).

IL-22 and IL-22R1 expression in the human brainThe fact that IL-22 and its soluble antagonist receptor,IL-22BP, are differentially expressed in MS patients ascompared to HC suggests that this cytokine may be in-volved in the immunopathogenesis of MS. For this rea-son, we looked for putative target cells of IL-22 in theCNS. This cytokine is recognized through the IL-22 het-erodimeric receptor composed of IL-10R2 and IL-22R1subunits [44]. Of note, IL-10R2 can also bind IL-10,IL-26, IL-28(α/β), and IL-29, whereas IL-22R1 binds alsoIL-20 and IL-24 [30, 45], but only IL-22 is recognizedby these two subunits together [45]. IL-10R2 has beenpreviously shown to be expressed ubiquitously among tis-sues from hematopoietic and nonhematopoietic origins,whereas IL-22R1 expression was absent on hematopoieticcells [14, 41].Here, to explore whether human brain CNS cells ex-

press the IL-22 receptor and whether IL-22 is present inthe brain, we analyzed brain tissue sections from fiveMS cases and 18 non-MS controls. Using immunohisto-chemistry peroxidase staining, we aimed at detecting IL-22 and IL-22R1 on adjacent tissue sections with picturestaken at the exact same area for all samples (Fig. 2). Wewere able to detect IL-22 and IL-22R1 in the GM andthe WM of both control subjects (Fig. 2a) and MS pa-tients, either in areas deprived of lesions (Fig. 2b, c) orat the vicinity of plaques (Fig. 2d). Isotype controlsshowed that there was no unspecific staining neither ofthe primary nor of the secondary antibodies used inFig. 2 (Additional file 3: Figure S3). Of note, MS plaqueswere identified by MOG and HE staining (example isshown in Fig. 2d). The expression of the cytokine and itsreceptor was clearly stronger in MS than control tissue(Fig. 2b–d compared to Fig. 2a). Interestingly, themorphology of the IL-22- and IL-22R1-positive cellsclearly looked alike GFAP-positive astrocytes (Fig. 2c,arrow, first to third row), suggesting that the receptorand its cytokine were present on the same cells, for ex-ample see the two upper panels of Fig. 2c. The IL-22-and IL-22R1 expressing cells were often found in thevicinity of blood vessels. Since IL-22R1 was identified tobe expressed by endothelial cells in MS [22], we stainedfor Caveolin-1. However, we found that the staining of

IL-22 and its receptor reflected more the staining ofGFAP than the one of Caveolin-1 (Fig. 2c, d in particu-lar). Caveolin-1 delineates very nicely in blood vessels,whereas IL-22 and IL-22R1 are localized on fine pro-cesses around blood vessels and on cell bodies of stellatecells in proximity of blood vessels. Moreover, in MSlesions where strong astrogliosis occurs, IL-22 and IL-22R1 expressions was also enhanced (Fig. 2d).Next, using immunofluorescent confocal microscopy,

we wanted to determine whether IL-22 and IL-22 recep-tor indeed colocalize on the same cells. We also wantedto ascertain their expression on astrocytes as indicatedby light microscopy (Fig. 2). We found that IL-22 colo-calized with its receptor in the brain tissues of controlsubjects (Fig. 3a–d) as well as in the brain tissues of MSpatients (Fig. 4). In the brain of control subjects, the IL-22/IL-22R1 colocalization seemed to be slightly moreexpressed in the GM than in the WM (Fig. 3a, b),whereas in MS, it was clearly stronger in the plaques, ei-ther in the WM (Fig. 4a, b) or the subpial GM (Fig. 4c,d), than in the normal appearing white matter (NAWM)(Fig. 4a, b). We were also able to confirm that the IL-22/IL-22R1 couple was expressed on astrocytes in the brainof control subjects (Fig. 3a, c) as well as of MS patients(Fig. 4a, c). By contrast, co-staining of the IL-22/IL-22R1with Cav-1 was rarely detected (less than 1 % of Cav-1+

structures were also positive for IL-22/IL-22R1) (Fig. 3b).By analyzing adjacent slices of pictures taken at the verysame location, we noticed strong IL-22R1 expression inwhite matter plaques (Fig. 4a, b) as well as in subpial le-sions in gray matter (Fig. 4c, d).To ascertain the specificity of the antibodies used for

immunofluorescent confocal microscopy and to rule outautofluorescence, we performed isotype stainings, usingthe exact same protocol, acquisition parameters, and post-processing analysis methodology as for specific antibodies(Additional file 4: Figure S4 and Additional file 5: FigureS5). Of note, the pictures of the isotype controls weretaken at the very same location in the tissue slices as thepictures of the specific antibodies, except for Additionalfile 4: Figure S4c, which was not immediately adjacent toFig. 3c, d. Otherwise, tissue pictured in Additional file 4:Figure S4a, b was immediately adjacent to the one repre-sented in Fig. 3a, b; the same was true for Additional file 5:Figure S5a with Fig. 4a, b and Additional file 5: Figure S5bwith Fig. 4c, d.

Fig. 2 IL-22 and IL-22R1 are expressed in human brain. IL-22, IL-22R1, GFAP, and Caveolin-1 immunohistochemistry peroxidase stainings of braintissue sections of control (a) and MS (b–d) patients. MOG and HE stainings were used to detect MS demyelinating plaques (d). All four picturesbelonging to one column (a, b, c, or d) were always immediately adjacent to each other. Pictures a, b, and c were taken at areas at the borderbetween GM and NAWM, whereas pictures in d were taken from the same location at the edge between NAWM and a plaque. Inserts in columnsa and b represent a threefold magnification of the selected area. Arrow: astrocyte-like pattern. a study patient B-C2, b and d study patient B-MS3,c study patient B-MS5 (Table 2). Scale bar, 50 μm (a, b: ×20, c, d ×40). GM: gray matter, NAWM: normal appearing white matter, WM: white matter.Representative pictures obtained from the observations of seven control and five MS autopsy samples

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Fig. 3 Specific expression of IL-22 and its receptor, IL-22R1, by astrocytes in the brains of control patients without MS and suffering from otherneurological diseases. Laser scanning confocal microscopy observations were performed in brain tissue autopsies (Basel cohort). Brain autopsylabeled for IL-22 (first panel, green), IL-22R1 (second panel, red), and Caveolin-1 or GFAP (third panel, blue). Merged images are depicted as compositeimages in the lower panel (colocalization of IL-22R1 and GFAP appears in yellow and triple colocalization of IL-22, IL-22R1, and GFAP in white). Insertsare a ×10 zoom of the selected area (a, b, lowest panels). Images a and b were taken on autopsied brain tissue from control patient B-C6 and c and dfrom control patient B-C1 (Table 2). Arrows in a point at astrocytes, and stars in b at blood vessels. Bars, 50 μm. GM: gray matter, WM: white matter.Representative pictures obtained from the observations of seven control autopsy samples

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Additional staining performed on biopsies from con-trol patient L-C5 confirmed that there was a very strongcolocalization of IL-22R1 with GFAP (Additional file 6:Figure S6a) but not with Cav-1 (Additional file 6: FigureS6b, c) or with MAP-2 (Additional file 6: Figure S6c).Staining with anti-VWF, an alternative blood vesselmarker, led to the same results as with anti-Cav-1, i.e.,no colocalization with IL-22R1 (Additional file 7: FigureS7). Interestingly, IL-22R1 expression was particularlyhigh in the close vicinity of blood vessels, lining them

up. However, we could show that this proximity was notdue to endothelial expression of IL-22R1 (Additionalfile 6: Figure S6b) but was rather attributable to expres-sion by astrocytic feet. Of note, to ascertain that the de-tection of IL-22R1 used so far indeed indicated theexpression of the whole IL-22 receptor, we assessed theexpression of the other subunit of the IL-22 receptor,i.e., IL-10R2, and found that, indeed, IL10R2 colocalizedwith IL-22R1, clearly establishing that the heterodimericIL-22 receptor complex is fully expressed in the CNS

Fig. 4 Strong expression of IL-22 and its receptor, IL-22R1, in the plaques of MS brains. Immunofluorescence of human brain tissue sectionsstained for IL-22 (green), IL-22R1 (red), and Cav-1 or GFAP (blue) in MS brain tissue autopsies. Images were taken from patient B-MS3 patient(Table 2) in a plaque located in the subcortical WM (a, b) and in a plaque located in the subpial GM (c, d). The lowest panels depict a ×5magnification of the white squares displayed in the above panels. Arrow: astrocytes, star: blood vessels. Arrowheads point at triple IL-22/IL-22R1/GFAP colocalization (a, c). Bars, 50 μm. NAWM: normal appearing white matter. Representative pictures obtained from the observations of five MSautopsy samples

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(Additional file 6: Figure S6d). Altogether, this set ofexperiments establishes that the IL-22 receptor isexpressed in the human brain, mostly on astrocytes, andthat IL-22 colocalize with its receptor. We also demon-strate that even if the IL-22/IL-22R1 couple is present inthe brain of control patients with other neurological dis-ease than MS, it nevertheless predominates in the brainof MS patients, in particular in the plaques.

IL-22 increased the survival of astrocytesHaving shown that the IL-22 receptor was expressed inthe human brain, predominantly by astrocytes, we decidedto investigate the role of IL-22 on astrocytes. To this end,we selected human primary astrocytes. The purity of thoseprimary astrocytes, as assessed by GFAP staining on flowcytometry, was close to 80 % (Additional file 8: Figure S8).Such as shown by flow cytometry (Fig. 5a) and by im-munofluorescence (Fig. 5b–d), these astrocytes expressedboth subunits of the IL-22 receptor, confirming that IL-22could bind to these cells.In order to determine whether IL-22 has a protective

or deleterious effect on astrocytes, we studied its impactin terms of cell death, such as measured by 7-AAD, amarker of cell viability.We found that IL-22 protected human astrocyte from

cell death. Indeed, nutriment-deprived astrocytes thathad been treated with IL-22 (plain blue line) exhibited asignificant higher survival than astrocytes cultured in thesame conditions but without IL-22 (dashed blue line)(Fig. 6a, c). Even if the improved survival of astrocytesattributable to IL-22 was relatively mild (increase of sur-vival 2.4 % on day 2, 7 % on day 3, and 4.2 % on day 5),it was significant and occurred during a time of the cul-ture (day 2, 3, and 5), which corresponded to a nadir interms of astrocyte survival (Fig. 6c, blue lines).Then, we assessed whether the protective function of

IL-22 on astrocytes was maintained in a still more hos-tile environment, i.e., under inflammatory conditions.Therefore, we compared TNFα-treated astrocytes withor without adding IL-22. We found that whereas therewas a steady and constant cell death of the TNFα-treated astrocytes population which had not received IL-22 (dashed red line) (Fig. 6b, c), there was a much bettersurvival of astrocytes co-treated with TNFα and IL-22(plain red line) from day 7 (Δ = 8.9 %) to day 9 (Δ =20.6 %) (Fig. 6b, c). Regarding TNFα- versus IL-22- andTNFα-treated human astrocyte conditions, TNFα-treated cells showed steady shrinkage of surviving cellnumber till the end of the kinetic (Fig. 6c). Addition ofIL-22 to the TNFα-treated cells drastically amelioratedcell survival from day 7 on, as reflected by the decreasedfrequency of 7-AAD-positive coupled with a rise of 7-AAD-negative cell frequency, i.e., more alive astrocytes.

In an attempt to determine if an anti-apoptotic mech-anism may account for the protective effect of IL-22, weassessed whether, among 7-AAD-negative living cells,there were cells in an early stage of apoptosis, such asrevealed by Annexin V staining. Gating on live cells only(7-AAD-negative), we found that the mean fluorescenceintensity (MFI) of Annexin V was significantly less in-tense in IL-22-treated cells than their untreated counter-parts on the first day of culture (27.1 % MFI difference),showing that the former was less apoptotic than thelatter (Fig. 6d). Furthermore, we found that IL-22 treat-ment significantly attenuated the pro-apoptotic effectof TNFα on day 9 of the assay (22.8 % MFI decrease,Fig. 6d).Finally, some authors have shown that IL-22 promotes

proliferation of epithelial cells [19]. Thus, we examinedwhether the pro-survival effect of IL-22 on astrocytescould be ascribed to this property of IL-22. However, wedid not find such an effect of IL-22 (data not shown).

DiscussionSo far, IL-22 has been barely studied in the context ofMS. Possible reasons may include the unchanged courseof EAE in mice deficient for IL-22 as compared to wild-type mice [46] and also the fact that this cytokine doesnot target immune cells [14, 21, 41]. Yet, there are someelements suggesting that this cytokine may be involved inthe immunopathogenesis of MS. Indeed, a polymorphismof the gene coding for interleukin-22 binding protein be-tween MS patients and controls has been described re-cently [32]. Interestingly, in EAE, IL-22BP knock-out micehave a decreased neuroinflammatory profile and an overallless severe disease course as compared to wild-type litter-mates, strongly suggesting that IL-22 attenuates diseaseseverity [35].We found that the level of IL-22 was higher in the

serum of MS patients than HC (Fig. 1a). In fact, this in-crease was entirely attributable to MS patients with ac-tive disease (Fig. 1b). This increase of serum IL-22seems to be attributable to an increased secretion of thiscytokine by PBMC (Fig. 1d, e), in particular CD4+ T cells(Additional file 1: Figure S1) [41]. Similarly to us, othersrecently found that CD4+ T cells, and more specificallyTh17 and Th22 cells, of MS and neuromyelitis optica(NMO) patients secreted more IL-22 than those of HC[47, 48]. Our results are supported by findings in Lewisrats where the expression of IL-22 is increased duringthe acute phase of EAE and decreased in its recoveryphase, while IL-10 and IL-17 levels remain unchanged.These variations suggest that there is a tight correlationbetween this cytokine and the disease course [49]. Thus,in an attempt to understand the regulation of IL-22, weexamined IL-22BP, the soluble antagonist receptor of IL-22. Contrasting with IL-22, we saw no difference between

Fig. 5 Colocalization of both subunits of IL-22 receptor on primary astrocytes. a Flow cytometry experiments show the expression of bothsubunits of the IL-22 receptor, IL-10R2, and IL-22R1 (in red), on human primary astrocytes (HA) as compared to their isotype control counterparts(in black). b–d By immunofluorescent confocal microscopy, there is a colocalization of GFAP (green) with IL-10R2 (red) (b) and with IL-22R1 (red)(c) as well as of IL-10R2 (green) with IL-22R1 (red) (d). The colocalization appears is depicted in yellow (arrows, MERGE). DAPI is represented in blueand not included in MERGE overlay. Bars, 50 μm

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MS patients and HC at the protein level. However, wefound that mRNA coding for IL-22BP was mainly pro-duced by monocytes and moDCs (Additional file 2: FigureS2), corroborating what has recently been described inmice and humans [35, 43]. We then found that the level

of IL-22BP coding mRNA was higher in the monocytesand moDCs of MS patients than HC (Fig. 1i, k). Neverthe-less, contrasting again with IL-22, this increase was notclearly ascribable to MS patients with active disease, evenif there was a trend (Fig. 1j). Altogether, these data suggest

Fig. 6 Enhanced survival of IL-22-treated primary astrocytes in insulting conditions. Primary astrocyte cells (HA) were cultured in 24-well platesstarting at day −1 and were treated every second day starting at day 0 with astrocyte medium (AM)—representing the optimal culture mediumfor astrocytes—or with RPMI (poor medium, hereafter referred to as untreated), with or without addition of the following: IL-22 alone, TNFα alone,IL-22 and TNFα together, or still staurosporine (STS) alone, the latter representing a potent inducer of apoptosis. Cells were stained with 7-AADand Annexin V and analyzed by flow cytometry to assess their survival and apoptotic status. Histograms represent 7-AAD profile of untreatedversus IL-22 (a) and of TNFα versus IL-22 + TNFα (b). Survival profile analysis was performed by flow cytometry by following the frequency ofliving cells (i.e., 7-AAD-negative cells, Fig. 6 c) and, among those that were not dead (7-AAD-negative cells), by following the proportion of thosesurviving cells but which underwent apoptosis (d). Each dot represents the median of six replicates, except for AM and STS conditions (threereplicates). Orange arrows indicate treatment renewal. Considering STS treatment, 7-AAD kinetic was stopped after 4 days as all recovered cellswere dead at this time point.*comparison of IL-22-treated versus untreated cells; †comparison of TNFα- versus TNFα + IL-22-treated cells. Thevertical bars determine the 75th percentile of the median. Significance was calculated with unpaired non-parametric Mann–Whitney test.* or †P < 0.05, ** or ††P < 0.01

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that IL-22 is under tight control of IL-22BP, such as it isthe case for instance for the control of IL-1β by IL-1RA[50]. Yet, during a relapse, IL-22 seems to “overrule” thiscontrol, such as revealed by the significant increase of IL-22 in the serum of those patients.Somewhat contrasting with the data in peripheral

blood, we found that, in the CSF of active MS patients,IL-22BP (Fig. 1h), but not IL-22 (Fig. 1c), was detectable.The absence of IL-22 in the CSF of active MS patients(Fig. 1c) may simply indicate that IL-22 plays no role atthe CNS level. However, we do not think that this obser-vation should lead to such conclusion. First, it is well

established that the absence of a cytokine in the CSFdoes not preclude a paramount role in CNS inflamma-tion, such as reflected, for instance, by IL-6 [32, 51]. Sec-ond, and more important, since IL-22BP was present inthe CSF of active MS patients (Fig. 1h), it is tempting tohypothesize that it is in reaction to its ligand, i.e., IL-22.Third, confirming the results of others [14], we were notable to detect IL-22R1 on hematopoietic cells (data notshown), further pointing to the rationale to search forother target cells, in this case, the brain.Thus, we examined whether brain cells did express IL-

22 receptor. Whereas IL-10R2 is more or less ubiquitous

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[14], the expression of IL-22R1 is much more re-stricted. Therefore, having shown that both subunits ofIL-22 receptor colocalized on astrocytes (Additional file 6:Figure S6d), we thereafter focused on the more restrictivesubunit IL-22R1. A major finding was the expression ofIL-22R1 on astrocytes of both control and MS patientsbut clearly predominating in the latter. In particular, thisexpression was high in MS plaques or adjacent to bloodvessels, pointing to a wide expression in perivascularastrocyte end feet (Fig. 4). Such as Kebir et al., we ob-served colocalization of IL-22R1+ and caveolin-1+, indicat-ing an expression of this receptor by endothelial cells [22];but in our hands, this expression was restricted to thebrain of non-MS patients and in the NAWM of one MSpatient and was of limited extent. We could also rule outa constitutive expression of IL-22R1 by neurons since wenever observed MAP-2+ and IL-22R1+ colocalization(Additional file 6: Figure S6c).The IL-22 cytokine was shown to be expressed in the

brain and spinal cord of mice [52, 53]. Having demon-strated that human astrocytes expressed high levels ofthe IL-22 receptor, we subsequently looked for IL-22presence in the CNS. We found that this cytokine wasindeed present and that it colocalized with IL-22 recep-tor on astrocytes, further suggesting that IL-22 targetsastrocytes. However, whether the detected IL-22 was oflymphocytic origin or whether it was produced by resi-dent cells of the CNS, for instance astrocytes themselvesin an autocrine fashion, remains to be determined. Thefact that IL-22 was not detected in the CSF somewhatargues for a production by CNS resident cells ratherthan a secretion by T cells. Nevertheless, one shouldnot forget that IL-22 in the CSF may also be trapped byIL-22BP, since, in our hands, the latter was detected inthe CSF.Astrocytes are crucial to maintain CNS homeostasis and

are now recognized to play a role in the pathogenesis ofautoimmune demyelinating diseases. In NMO, a CNS dis-ease sharing many features with MS, astrocytes play acentral role as they are the cells expressing aquaporin-4(AQP4), a water channel embodying the antigen againstwhich the autoimmunity of the NMO antibodies is di-rected [54, 55]. In MS, astrocytes have been increasinglyrecognized as being an important component of thepathogenesis of the disease [56, 57]. KIR-4.1, which wasrecently found to be a possible target of auto-antibodies inhuman MS, is also expressed by oligodendrocytes and as-trocytes [58]. To explore what could be the effect of IL-22on astrocytes, we resorted to primary human astrocytes.We could confirm that these cells had astrocyte-characteristics and harbored both IL-22 receptor subunits(Fig. 5). We found that IL-22-treated astrocytes exhibitedan increased survival as compared to untreated ones, and,interestingly, this effect was maintained in inflammatory

conditions since IL-22 mitigated the effect of TNFα. Thiseffect was mediated, at least in part, by a decrease of apop-tosis. Supporting these findings, previous studies haveshown that IL-22-treated rat pheochromocytoma cellsexhibit a modest increased survival in serum-deprivedconditions [59]. Some authors have found that IL-22 in-creased the proliferative function of keratinocytes [19, 60]or colonic epithelial cells [42]. While we could reproducethese data on HaCaT keratinocyte cell line, we did not ob-serve any proliferative effect of IL-22 on primary humanastrocytes (data not shown), further pointing to a pro-survival effect of IL-22 on existing astrocytes. Furtherstudies investigating by which mechanism(s) IL-22 modu-lates astrocyte survival are warranted to better understandthe role of this cytokine on its newly defined CNS target.

ConclusionsIn conclusion, we have shown (i) that MS patients in re-lapse harbor significantly higher serum levels of IL-22, (ii)that astrocytes express the IL-22 receptor, (iii) that there isa colocalization of IL-22 with astrocytes, and (iv) that IL-22 has pro-survival properties on primary human astro-cytes. Astrocytes can play a dual role when challenged,depending on the nature of the insult, harboring either abeneficial or a detrimental phenotype [57]. Thus, havingshown that IL-22 is a player in the immunopathogenesisof MS, we will now examine whether the net effect of IL-22 on astrocytes is pro- or anti-inflammatory.

Additional files

Additional file 1: Figure S1. Different leukocyte subtypes are able toproduce and release IL-22 upon stimulation. Total PBMC were isolatedand MACS-sorted into monocytes (CD14+), B cells (CD19+), CD4+ T cells,CD8+ T cells, and NK cells (CD56+). Cells were either treated with SEB,R848, or CD3/CD28 beads (CD3/28) for 18 h, PMA/ionomycin (PMA/iono)for 6 h, or left to rest for 18 h (unstim). Upon stimulation, all leukocyteswere able to secrete significant amount of IL-22. Nevertheless, CD4+ Tcells represented the major IL-22 source. Except for R848, all polyclonalstimulations induced similar level of secreted IL-22 in each respective cellsubpopulation. Boxes represent the median, and bars the 75th percentile.n = 5 study subjects.

Additional file 2: Figure S2. Monocytes and moDCs are the mainIL-22BP-expressing cells. Total PBMC were isolated and MACS-sorted intomonocytes (CD14+), B cells (CD19+), CD4+ T cells, CD8+ T cells, NK cells(CD56+), and moDCs. Cells were either directly processed or treated for6 h with PMA/ionomycin (PMA/iono) or stimulated for 18 h with SEB aftersorting. Expression level is relative to 18S ribosomal RNA housekeepinggene.

Additional file 3: Figure S3. Peroxidase stainings are specific for IL-22,IL-22R1, GFAP, and Caveolin-1 in autoptic brain tissue from control andMS subjects. Immunohistochemistry peroxidase stainings of isotypecontrols of goat anti-IL-22, mouse anti-IL-22R1, rabbit anti-GFAP, andrabbit anti-Cav-1 antibodies. Pictures of A, B, C, and D have been acquiredat the exact same location as for specific antibodies and mirror exactlypanels A, B, C, and D, respectively of Fig. 2. Protocol and images wereprocessed exactly the same way for specific antibodies as for isotype controls.Background is negative for goat and mouse isotype controls (respectivelyIL-22 and IL-22R1 in Fig. 2). Background for the rabbit isotype control wassomehow higher, especially in blood vessels, but remained unspecific and

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thus did not compromise GFAP and Cavolin-1 specificity. A: study patientB-C2, B and D: study patient B-MS3, C: study patient B-MS5 (Table 2). Scalebar, 50 μm (A–B, ×20; C–D, ×40). GM: gray matter, NAWM: normal appearingwhite matter, WM: white matter. Representative pictures obtained from theobservations of seven control and five MS autopsy samples.

Additional file 4: Figure S4. Immunofluorescence stainings are specificfor IL-22, IL-22R1, GFAP and Caveolin-1 in autoptic brain tissue fromcontrol patients. Immunofluorescence staining of isotype controls ofgoat anti-IL-22, mouse anti-IL-22R1, rabbit anti-GFAP and rabbit anti-Cav-1antibodies. Panel A for A, B for B, and C for C and D, respectively, areduplicates of images of Fig. 3 representing the same tissue location in thebrain. Few unspecific background/autofluorescence could be observed,likely due to tissue quality (brain autopsies). The settings of isotype controlexperiments were exactly the same as for specific antibodies experiments(acquisition parameters and post-processing analyses). Images from panelA and B were taken from B–C6 and C from B–C1 control patient autopsybrain tissues, as for Fig. 3 (Table 2). Bars, 50 μm. GM: gray matter, WM: whitematter. Representative pictures obtained from the observations of sevencontrol MS autopsy samples.

Additional file 5: Figure S5. Immunofluorescence stainings are specificfor IL-22, IL-22R1, GFAP and Caveolin-1 in autoptic brain tissue from MSpatients. Immunofluorescence staining of isotype controls of goat anti-IL-22, mouse anti-IL-22R1, rabbit anti-GFAP, and rabbit anti-Cav-1 antibodies.Panel A for A and B, and B for C and D, respectively, mirror exactly imagesin Fig. 4, being from the exact same location and processed identically(same acquisition parameters and post-processing analyses). Thin unspecificautofluorescent area in 488 and 564 nm could be observed. Pictures weretaken in patient B-MS3 brain section, again, as for Fig. 4 (Table 2). Bars, 50μm. GM: gray matter, NAWM: normal appearing white matter, WM: whitematter. Representative pictures obtained from the observations of five MSautopsy samples.

Additional file 6: Figure S6. Colocalization of GFAP and IL-10R2, butnot Cav-1 and MAP-2 with IL-22R1 in the brain. Laser scanning confocalmicroscopy of brain biopsies labeled for IL-22R1 (red) in associationwith: A) the astrocytic marker, GFAP (green), B) the caveolin-1 endothelialmarker (green), C) the caveolin-1 endothelial marker (white) and theneuronal marker, MAP-2 (green), and D) the IL-10R2 subunit receptormarker (green). DAPI staining (blue) is represented in inserts on the upperleft side. Images were taken on biopsied brain tissue from control patientL-C5 (A to C) and L-C7 (D) (Table 2). Bar, 50 μm. Representative picturesobtained from the observations of 11 control biopsy samples.

Additional file 7: Figure S7. There is a high degree of colocalizationbetween von Willebrand factor and Caveolin-1, but not between IL-22R1and any of these two endothelial cell markers in the human brain.Immunofluorescence confocal microscopy images of VWF (blue), IL-22R1(red), and Cav-1 (green) of two slides, taken at different parts of the brain,of L-C5 study subject (Table 2) are depicted (A and B). Counterstainingwas performed with DAPI. Bars, 50 μm.

Additional file 8: Figure S8. Large majority of GFAP-positive cells inHA primary astrocytes. Flow cytometry histogram depicting GFAP stainedHA primary astrocytes (the bar indicates GFAP-positive cells, in percent).

Abbreviations7-AAD: 7-aminoactinomycin D; AM: astrocyte medium; AQP4: aquaporin-4;BSA: bovine serum albumin; Cav-1: caveolin-1; CFS: cerebrospinal fluid;CFSE: carboxyfluorescein succinimidyl ester; CIS: clinically isolated syndrome;CNS: central nervous system; DAPI: 4′,6-diamidino-2-phenylindole;EAE: experimental autoimmune encephalomyelitis; FCS: fetal calf serum;GFAP: glial fibrillary acidic protein; GM: gray matter; GM-CSF: granulocytemacrophages colony-stimulating factor; HA: human astrocytes; HC: healthycontrol; HE: hematoxylin and eosin; IL: interleukin; IL-22BP: interleukin-22binding protein; Iono: ionomycin; MAP-2: microtubule-associated protein 2;MCAM: melanoma cell adhesion molecule; MFI: mean fluorescence intensity;moDCs: monocyte-derived dendritic cells; MOG: myelin oligodendrocyteglycoprotein; MS: multiple sclerosis; NAWM: normal appearing white matter;NK cell: natural killer cell; NMO: neuromyelitis optica; PBMC: peripheral bloodmononuclear cells; PBS: phosphate-buffered saline; PMA: phorbol myristateacetate; PP-MS: primary progressive multiple sclerosis; R848: resiquimod;RR-MS: relapsing remitting multiple sclerosis; RT: room temperature;

SEB: staphylococcal enterotoxin B; SLE: systemic lupus erythomatosus;SP-MS: secondary progressive multiple sclerosis; Th cell: T helper cell;TNF: tumor necrosis factor; VWF: von Willebrand factor; WM: white matter.

Competing interestsMyriam Schluep has served as a consultant for Merck Serono and hasreceived honoraria, payment for development of educational presentationsand travel support from Merck Serono, Biogen Dompé, Novartis, Sanofi-Aventis and Bayer Schering.Renaud Du Pasquier has served on scientific advisory boards for Biogen Idec,Merck Serono, Teva, and Novartis and has received funding for travel orspeaker honoraria from Abbvie, Biogen Idec, Teva, Merck Serono, and BayerSchering Pharma.All other authors declare that they have no competing interests.

Authors’ contributionsGP, AM, and RDP made the experimental conception and design. GP, LE, AM,MC, and MG performed the manipulations and experiments. GP, AM, LE,RDP, and NSW helped in the data analysis. MS, NSW, and RDP contributedthe reagent/material/biological sample/equipment. GP, RDP, AM, and NSWwrote the paper. All authors read and approved the final manuscript.

Authors’ informationThis work is the result of the PhD thesis by GP.RDP is head of the laboratory of Neuroimmunology in the Service ofNeurology at the University hospital of Lausanne (CHUV), Switzerland. Hislaboratory has long standing expertise in characterizing immune responsesin an MS context. His laboratory has facilitated access to MS-patient biologicalsamples (CSF, PBMC, serum), thanks to the collaboration with Dr MyriamSchluep (MS).NSW is head of the laboratory of Neurobiology in the Department ofBiomedicine and Neurology, at the University hospital of Basel, Switzerland.She is an expert in the field of MS neurobiology and provided crucial help tothe processing and analysis of the highly valuable MS autopsied braintissues.

AcknowledgementsWe thank G. Le Goff for the invaluable help in enrolling patients andobtaining blood samples. We are also indebted to Dr Jean-François Brunetand Dr Jocelyn Bloch for providing us with the brain tissue taken fromneurosurgical biopsies.This work was supported by the Swiss MS Society and by a subsidy forresearch 320030_138411 from the Swiss National Foundation to RDP. NSWand MG have been supported by the National Multiple Sclerosis Society ofthe United States of America (grant RG 4249A2/2). We are grateful to the UKMultiple Sclerosis Tissue Bank.

Author details1Laboratory of Neuroimmunology, Center of Research in Neurosciences,Department of Clinical Neurosciences and Service of Immunology andAllergy, Department of Medicine, CHUV, 1011 Lausanne, Switzerland. 2Serviceof Neurology, Department of Clinical Neurosciences, CHUV BH-10/131, 46,rue du Bugnon, 1011 Lausanne, Switzerland. 3Neurobiology, Department ofBiomedicine, University Hospital Basel, University of Basel, 4031 Basel,Switzerland.

Received: 5 February 2015 Accepted: 3 June 2015

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